Base Composition Studies

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Chapter 9

• Part 2

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Base Composition Studies

• Chargaff used chromatographic methods to separate
  and determine the 4 bases
• wanted to determine the amount of each in various
  organisms
• Conclusions
• A amount is proportional to T and C to G
• A G T C, purine to pyrimidine is equal
• percentage of (CG) is not equal to percentage of
  (AT) varies by organism
• Disproves the tetranucleotide theory

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X-Ray Diffraction

• Bombard a molecule with x-ray that will scatter
  in a pattern based on location of atoms
• Rosalind Franklins x-ray picture of DNA
• must be a helix with 0.34 nm periodicity

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Watson-Crick Model

• Published in 1953 double-helix for DNA
• Key points about the model
• 2 strands coil to the right around a central axis
• anti-parallel chains
• nitrogenous bases are flat and perpendicular to
  axis stacked above each other
• nitrogenous bases of opposite chain paired, A to
  T and G to C complementarity important for DNA
  replication and gene expression
• each turn is 3.4 nm long
• larger major groove and smaller minor groove
• double-helix is 2 nm in diameter
• Blue is the most important parts of the model

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Additional DNA Points

• Sugar-phosphate backbone is hydrophilic and it
  functions to protect the hydrophobic nitrogenous
  bases in the interior
• helps stabilize the helical structure
• 2 small errors in the model
• actually 10.4 bp per turn not 10
• 34.6 rotation not 36

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Predictions Made by Watson-Crick

• DNA will replicate in a semiconservative fashion
  consist of one old strand and one new strand
• Storage of genetic information is in sequence of
  bases
• Mutation/genetic changes would result from
  alteration of base

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Alternative Form of DNA Exist

• Conformational forms of DNA
• A-DNA high salt conditions, more compact
  probably not in vivo
• B-DNA aqueous, low salt conditions
  biologically significant form
• C-, D-, E-, and P-DNA forms
• P-DNA is named after Linus Pauling, artificial
  stretching of DNA, longer, narrower and PO4 on
  the inside
• Z-DNA synthetic fragment of G-C, made a
  left-handed helix with zigzag conformation, not
  sure if in vivo

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• Use Svedberg Coefficient (S) to determine the
  size of the RNA molecules, bigger S the larger
  the molecular mass, also affected by size and
  shape

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Unique RNAs

• Small nuclear RNA (snRNA) process mRNA
• Telomerase RNA DNA replication at ends of
  chromosome
• Antisense RNA, microRNA (miRNA), short
  interfering RNA (siRNA) all part of gene
  regulation
• DNA stores information and RNA helps express that
  information

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Melting Profiles

• Isolated DNA if heated will denature, slowly
  allow to cool, the strands will reassociate, also
  do with complementary sequences from different
  species
• Unwinding DNA causes the viscosity to decrease
  and the UV absorption to increase hyperchromic
  shift
• Melting profile vs temperature midpoint of each
  curves is melting temperature (Tm)
• higher the Tm the more G-C,
• lower the Tm the more A-T

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Molecular Hybridization Techniques

• Denature/renature hybridization studies
• Works with 2 or more different sources if
  complementary bases present
• Also works with DNA and RNA
• in situ hybridization cells on a slide-either
  mitotic or interphase treat to hybridization
  technique (open DNA)
• Use ssDNA or RNA as probe

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Reassociation Time

• Large genomes, the longer it takes to associate
• Some DNA has repetitive sequences reassociates
  more quickly than stretches of unique sequences
• Repetitive sequences go from short and repeated
  millions of times, or longer and few repeats or
  levels in between the extremes

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Electrophoresis

• Separate by size and charge using an electric
  field
• Use polyacrylamide or agarose as the matrix
• Large molecules move slower than small fragments
• Beginning to Southern/Northern, DNA sequencing
  methods, etc